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Award Number: W81XWH-11-1-0113 


AD 


TITLE: DHHC3 contributions to breast cancer 


PRINCIPAL INVESTIGATOR: Michael Freeman 


CONTRACTING ORGANIZATION: Children's Hospital Corporation 
Boston, MA 02115 


REPORT DATE: September 2013 


TYPE OF REPORT: Annual 


PREPARED FOR: U.S. Army Medical Research and Materiel Command 
Fort Detrick, Maryland 21702-5012 


DISTRIBUTION STATEMENT: Approved for Public Release; 

Distribution Unlimited 


The views, opinions and/or findings contained in this report are those of the author(s) and 
should not be construed as an official Department of the Army position, policy or decision 
unless so designated by other documentation. 



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1. REPORT DATE 2. REPORT TYPE 

September 2013 Annual 

3. DATES COVERED 

1 September 2012 - 31 August 2013 

4. TITLE AND SUBTITLE 

DHHC3 contributions to breast cancer 

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W81XWH-11-1-0113 

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6. AUTHOR(S) 

Michael Freeman 

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Children's Hospital Corporation 

Boston, MA 02115 

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U.S. Army Medical Research and Materiel Command 

Fort Detrick, Maryland 21702-5012 

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Approved for Public Release; Distribution Unlimited 

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14. ABSTRACT 

Palmitoylation is an important post-translational modification that plays a critical role in regulating protein localization, activity, 
and stability, as well as multiprotein complex formation. Our preliminary data suggested that DHHC3, a palmitoylating 
enzyme, plays a critical role in breast cancer growth, invasion, and/or metastasis, at least partly by palmitoylating certain 
proteins resident in tetrapanin-enriched microdomains. However, very few proteins have been identified as DHHC3 subtrates. 
Thus, we proposed to comprehensively identify DHHC3 subtrates in breast cancer by integrating our palmitoyl protein 
identification and site characterization method with triplex SILAC. We established this multiplexed quantitative palmitoyl- 
proteomics method in year 1. Here, we applied this multiplexed quantitative palmitoyl-proteomics method to identify DHHC3 
substrates in breast cancer MDA-MB 231 cells. We identified over 680 candidate palmioyl proteins, among 70 candidate 
palmitoyl proteins, among which 70 are candidate DHHC3 subtrates. 

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Table of Contents 


Page 


Introduction..... 4 

Body.4 

Key Research Accomplishments..... 6 

Reportable Outcomes... 8 

Conclusion...8 

References. 8 


Appendices 


3 










Introduction 


Tetraspanin-enriched microdomains (TEMs) are critical signaling platforms, playing a 
key role in cancer progression [1, 2]. Many TEM-resident proteins, including laminin-binding 
integrins, tetraspanins, and their partner proteins, are regulated by post-translational 
palmitoylation. Protein palmitoylation, the only reversible lipid modification, is critical for the 
regulation of protein localization, activity, and stability as well as multiprotein complex 
formation [3], Palmitoylation in human is carried out by 23 DHHC enzymes [4], Dr. Martin 
Hemler’s lab showed that the ablation of DHHC3 not only inhibits the palmitoylation of integrin 
a6 and p4 subunits [5], but also disrupts TEMs and markedly alters cell morphology, invasion 
and signaling through focal adhesion kinase (FAK) in breast cancer cell lines. The results 
strongly suggested that DHHC3 plays a critical role in breast cancer progression. Because the 23 
human DHHC proteins catalyze the palmitoylation of hundreds of cellular proteins, it is likely 
that at least dozens of proteins are palmitoylated by DHIIC3. However, little is known about 
which proteins, except integrin a6 and (14 subunits, are DHHC3 substrates in breast cancer cells. 

We developed a novel palmitoyl-proteomics method termed Palmitoyl Protein 
Identification and Site Characterization (PalmPISC) [6]. In this project, we proposed to 
comprehensively analyze DHHC3 substrates in breast cancer cells (Aim 3). We planned to 
integrate our PalmPISC method with stable isotope labeling by amino acids in cell culture 
(SILAC) [7], a very accurate and robust quantitative proteomics method, to identify known and 
novel DHHC3 substrates. In year 1, we successfully integrated our PalmPISC method with 
triplex SILAC labeling. Here, we applied the quantitative paimitoylproteomies method to 
comprehensively identify DIIHC3 substrates in breast cancer MDA-MB-231 cells. 


Body 


As shown in Figure 1, three populations of MDA-MB-231 cells were metabolically 
labeled with isotopically different SILAC amino acids in parallel. One group of control cells 
were cultured in “light” medium containing natural lysine (LysO) and arginine (ArgO), DIIHC3- 
knockdown cells were cultured in “heavy” medium containing )3 C 6 , i5 N 2 -lysine (Lys8) and 
I3 c 6 , !5 n 4 -arg inin e (ArglO), and the other group of control cells were cultured in “medium” 
medium containing 4,4,5,5-D.,-lysine (Lys4) and 13 C 6 -arginine (Arg6). After six doublings, when 
cellular proteins were at least 98% labeled with SILAC ammo acids, control cells labeled with 
LysO and ArgO and DHHC3-knockdown cells labeled with Lys8 and ArglO were mixed at 1:1 
ratio and palmitoyl proteins were isolated using our PalmPISC method, During the development 
of our PalmPISC method, we noticed that a small population of nonpalmitoylated proteins is 
always co-purified with palmitoyl proteins. Thus, to distinguish palmitoyl proteins from these 
contaminating proteins, we omitted hydroxylamine—a chemical provides selectivity for 
palmitoyl proteins—from our PalmPISC condition and isolated the contaminating proteins from 
control cells labeled with Lys4 and Arg6. Finally, we mixed the purified proteins together and 
performed in-depth quantitative proteomics analysis using GeLC-MS/MS and analyzed the 
SILAC dataset with MaxQuant (vl.O.13.13), a free software suite for SILAC data analysis [8]. 


4 






Membrane fraction 


PalmPISC 

hydroxylamlne(+) 


shCtrl 


Membrane fraction 

PalmPISC 
hydroxylamlnef 


Palmltoyl proteins 
& contaminating proteins 


Contaminating proteins 


GeLC-MS/MS 

Figure 1. Workflow for the identification of DHHC3 substrate in breast cancer. 


Theoretically, proteins that are palmitoylated by DHHC3 (i.e., DHHC3 substrates) will 
have a pattern of SILAC spectra shown in Fig. 2A, because the knockdown of DHHC3 reduces 
tiie palniitoylation level of its substrates while the omission of hydroxylamine prevents the 
purification of the substrates. In contrast, DHHC3 knockdown will not affect other palmitoylated 
proteins, thus these non-DHHC3-substrates will have a pattern shown in Fig. 2B. In addition, 
contaminating proteins will have a ratio of 1:1:1 (Fig. 2C), because DHHC3 knockdown or the 
presence/absence of hydroxylamine will not affect then purification. 


(A) 



(8) 



<C) 


£ 

VI 



£ 


1 

£ 

*A 










£ 



C 


H’ 

» 

£ 




. / 

m/i 



m/z 


m/z 


DHHC3-substrate 

Non-DHHC3-substrate 


Contaminating 


palmitoyl proteins 


palmitoyl proteins 


proteins 


Figure 2. Theoretic patterns of SfLAC spectra for (A) palmitoylated proteins that are DHHC3 substrates, (B) 
palmitoylated proteins that are not DHHC3 substrates, and (C) contaminating proteins. 


Our quantitive palmitoyl-proteomics analysis led to the identification of 1097 proteins 
with a false-discovery rate of 1%; among these proteins about 930 were quantitated (see Table 
SI). Using a cutoff value of 0.606 (p<0.05) for the “medium”/ “light” (M/L) SILAC ratio, we 
identified 687 candidate palmitoyl proteins. Moreover, using a cutoff value of 0.606 (p<0.05) for 
the “heavy”/ “light” (II/L) SILAC ratio, we identified 70 candidate palmitoyl proteins as 
candidate DHHC3 substrates (Table 1). Figure 3A showed a representive SILAC spectrum of a 
peptide derived from cytoskeleton-associated protein 4 (CKAP4), a known palmitoyl protein [3], 
DHHC3 knockdown led to the decrease of the palniitoylation level of CKAP4, suggesting that 


5 



CKAP4 is a candidate substrate of DHHC3. In contrast, as shown in Figure 3B, the 
pahnitoylation level of flotiilin-1 (FLOT1), also a known palmitoyl protein [4], was not affected 
by DF1HC3 knockdown, indicating that flotillin-1 is unlikely a DHHC3 substrate. 


M shCon 



CKAP4 

(B) shCon .hi i: US' ' 



FLOT1 


Figure 3: Repiehcnsivc SILAC spectra of a peptide derived from (A) a candidate DHHC3 substrate 
cytoskeleton-associatcd protein 4 (CKAP4) and (13) an unlikely DHHC3 substrate flotillin-1 (FLOT1). 


Table 1. List of candidate DHHC3 substrates 


Gene Name 

Protein Name 

Median/Light 

Ratio 

Heavy/Light 

Ratio 


Protein S100-A9 

0.160 


| SCAMP3 

Secretory carrier-associated membrane protei 


mkmbsi 


Sulfatase-modifying factor 2 




Membrane-associated progesterone receptor component 1 


0.280 


Catliepsin D 



NOL6 

Nucleolar protein 6 



LAMPT4A 

Lysosomal-associated transmembrane protein 4A 


0.340 

1 

CKLF-like MARVEL transmembrane domain-containing 
protein 3 

0.136 

0.345 

■ 

Heat shock protein beta-1 

jjWMlKm—ui 

0.369 

■ 

Integrin alpha-6 



TMEM192 

Transmembrane protein 192 

0.090 

0.380 

MREG 

Melauoregulin 

0.008 

0.380 

FAM108A1 

Abhydrolase domain-containing protein 

0.021 

0.396 

TMEM97 

Transmembrane protein 97;Protein MAC30 

0.155 

0.407 

NPC1 

Niemann-Pick Cl protein 

0.122 

0.409 

H1ST1H1C 

Histone HI.2 

0.553 

0.416 

CMTM6 

CKLF-like MARVEL transmembrane domain-containing 
protein 6 

0.222 

0.474 


6 
















TMEM179B 


OSTC 


BR13BP 


TMEM55A 

KIAA0090 

RFFL 


NFXL1 


TMED1 

STARD3NL 


IMP3 


GPX8 

M6PR 


PGRMC2 


EBUgmi 


ROMOl 

HSP90B1 


SLC4A7 


MFSD1 

BET) 


VKORC1 


SELI 


DERL2 

KTAA0754 


FAM36A 

SPCS1 


BANF1 


CDKAL1 


MRPL43 

SPRY2 


AUP1 


CKAP4 
MAN 1B1 
SAR I A 


PRDX4 


SOAT1 

SCARB1 

SPINT2 


DNAJA1 


LMF2 

RHOT2 

PCBP2 

TXN 


ZDHHC6 

STX7 

TPI1 

RAB27B 



_ Transmembrane protein 179B _ 0.0139 

Oligosaccharyltransferase complex subunit OSTC 0.293 


BRI3-binding protein 0,353 


Transmembrane protein 55A 0.100 


_ KIAA0090 _| 0.449 

_ E3 ubiquitin-protein ligase rififylin _ 

_ NF-Xl-type zinc finger protein NFXL1 __ 

_ Transmembrane emp24 domain-containing protein 1 _ 

_ MLN64 N-terminal domain homolog _ 

_ U3 small nucleolar ribomicleoprotein protein 1MP3 _ 

Probable glutathione peroxidase 8 


Cation-dependent mannose-6-phosphate receptor 


Membrane-associated progesterone receptor component 2 


l-acyl-sn-glycerol-3-phosphate acyltransferase alpha 


Vesicle-associated membrane protein-associated protein B/C 


Methyltransferase-like protein 7B 


Reactive oxygen species modulator 1 


_ Endoplasmin _ 

Solute carrier family 4 sodium bicarbonate cotransporter 
member 7 


Major facilitator superfamily domain-containing protein 1 

_ BET1 homolog _ 

Vitamin K epoxide reductase complex subunit 1 


Ethanolaminephosphotransferase 1 


Derlin-2 


Uncharacterized protein KIAA0754 


cDNA FLJ32471 fis, clone SKNMC2000322, highly similar to 
Peptidyl-tRNA hydrolase 2, mitochondrial (EC 3.1.1.29) 
Protein FAM36A 


Signal peptidase complex subunit 1 


_ Barrier-to-autointegration factor _ 

CDK5 regulatory subunit-associated protein 1-like 1 


Mitochondrial ribosomal protein L43 


_ Protein sprouty homolog 2 _ 

Ancient ubiquitous protein I 


Cytoskeleton-associated protein 4 


Endoplasmic reticulum mannosyl-oligosaccharide 1,2-alpha- 
mannosidase 


_ GTP-binding protein SARI a _ 

Peroxiredoxin-4 


Sterol O-acyltransferase 1 


Scavenger receptor class B member 1 


Kunitz-type protease inhibitor 2 


_DnaJ homolog subfamily A member 1 


Lipase maturation factor 2 


Mitochondrial RJio GTPase 2 


_ Poly(RC)-binding protein 2 isoform b variant _ 

_ Thioredoxin _ 

Sterol regulator)' element-binding protein cleavage-activating 

, • L/ • 13 o 

_ protein __ 

_ Probable palmitoyltransferase ZDHHC6 _ 0,0154 

_ Syntaxin-7 _ 0.109 

_ Triosephosphate isomerase _ 0.549 

Ras-related protein Rab-27B 0.539 



0 

.446 

0 

.223 

0 

.129 

0 

.117 

0 

.389 

0 

.595 

0 

.352 

0 

.493 

0 

.144 

0 

.416 

0 

.221 

\mE 

259 

IH 

284 

0 

456 

0 

169 

0 

046 

0 

025 

0 

021 

0 

546 

0 

572 

0 

301 

0 

079 

























































































DNAJCll 

DnaJ homolog subfamily C member 11 

0.332 

0.602 

ERGIC3 

Endoplasmic reticulum-Golgi intermediate compartment protein 

3 

0.050 

0.603 


Key Research Accomplishments 

1. Identification of about 700 candidate palmitoyl proteins from breast cancer MDA-MB- 
231 cells. 

2. Identification of 70 candidate DHHC3 substrates. 


Reportable Outcome 


Conclusion 

In summary, by integrating RNAi, SILAC, and PalmPISC, we developed a powerful tool 
for rapid identification of substrates for an individual palmitoyl acyltransferase. By using this 
quantitative palmitoyl-proteomics method, we identified integrin a6, a known DIIIIC3 substrate, 
and 69 novel candidate DHIIC3 substrates. 


References 

1. I-Iemler, M.E., Tetrcispanin functions and associated microdomains. Nat Rev Mol Cell 
Biol, 2005. 6(10): p. 801-1L 

2. Richardson, M.M., L.K. Jennings, and X.A. Zhang, Telraspanins and tumor progression. 
Clin Exp Metastasis, 2011. 28(3): p. 261-70. 

3. Salaun, C,, J. Greaves, and L.H. Chamberlain, The intracellular dynamic of protein 
palmitoylalion. J Cell Biol, 2010. 191(7): p. 1229-38. 

4. Korycka, J., et a!., Human DHHC proteins: a spotlight on the hidden player of 
palmitoylation. Eur J Cell Biol, 2012. 91(2): p, 107-17. 

5. Sharma, C., I. Rabinovitz, and M.E. Hemler, Palmitoylation by DHHC3 is critical for the 
function, expression, and stability of integrin alpha6beta4. Cell Mol Life Sci, 2012. 
69(13): p. 2233-44. 

6 . Yang, W., et al,, Proteome scale characterization of human S-acylated proteins in lipid 
rcift-enriched and non-raft membranes. Mol Cell Proteomics, 2010. 9(1): p. 54-70. 

7. Ong, S.E., et al., Stable isotope labeling by amino acids in cell culture, SILAC, as a 
simple and accurate approach to expression proteomics. Mol Cell Proteomics, 2002. 
1(5): p. 376-86. 

8 . Cox, J. and M. Mann, MaxQuant enables high peptide identification rates, individualized 
p.p.b.-range mass accuracies andproteome-wide protein quantification. Nat Biotechnol, 
2008. 26(12): p. 1367-72. 


8